title: High-throughput fluorescence polarization assay of ligand binding to IP3 receptors.
abstract: Fluorescence polarization (FP) allows quantification of the binding of a small fluorescent ligand to a larger protein because the free ligand rotates more rapidly than the bound form. This protocol describes an FP assay for the binding of fluorescein-labeled inositol 1,4,5-trisphosphate (IP3) to amino-terminal fragments of the IP3 receptor at different temperatures and in the presence of competing ligands. The method requires fluorescein-labeled IP3 and a plate-reader capable of FP measurements. The assay can measure low-affinity interactions in real time, it avoids use of radioactive materials, is nondestructive, and can resolve changes in Gibbs free energy (ΔG°), enthalpy (ΔH°), and entropy (ΔS°) that occur with ligand binding. It is applicable to any purified protein for which a fluorescent ligand is available. After optimization, the procedure can be completed in 1-6 h.
authors.affiliation: Department of Pharmacology, University of Cambridge, Cambridge CB2 1PD, United Kingdom.
authors.forename: Ana M
authors.initials: AM
authors.lastname: Rossi
authors.forename: Colin W
authors.initials: CW
authors.lastname: Taylor
mesh_terms: D003584:Cytological Techniques / Q000379:methods*; D005454:Fluorescence Polarization / Q000379:methods*; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000378:metabolism*; D008024:Ligands; D011485:Protein Binding; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D053496:Inositol 1,4,5-Trisphosphate Receptors; D008024:Ligands; D015544:Inositol 1,4,5-Trisphosphate